Hydra Biosciences, Inc. will develop a novel class of protein therapies for regeneration of heart tissue following acute myocardial infarction. This work is based on the discovery that vertebrates can regenerate heart muscle by inducing cardiac myocyte dedifferentiation and proliferation. Triggering myocyte proliferation after cardiac injury will enhance regeneration and reduce scar formation in man, but a specific cardiac mitogen has not been purified. Recently, published work has defined the existence of specific fetal cardiac mitogens in conditioned media of epicardial cells. We have demonstrated that neonatal cardiac myocytes proliferate in response to fetal calf serum and specific growth factors. Cells isolated from adult myocardium undergo mitosis in response to these unpurified mitogens. Hydra Biosciences will industralize the purification and cloning of neonatal cardiac mitogen, and test this mitogen in adult cardiac myocardium. The Specific Aims are: AIM 1. DEVELOP QUANTITATIVE, IN VITRO CARDIAC MYOCYTE PROLIFERATION ASSAYS. First, we will establish high-throughput neonatal cardiac myocyte proliferation assays using BrdU, H3P and survivin immunohistochemistry. Next we will establish a 96-well assay that can be automatically scored. Finally, we will establish adult cardiac myocyte proliferation assays. These assays will be used to demonstrate that mitogen purified using neonatal assays is effective in adult myocardium. AIM 2. DEFINE SOURCES OF MAMMALIAN CARDIAC MITOGEN. First, we will screen candidate mitogens implicated in embryonic cardiac development. These factors will be useful controls for subsequent studies. Second, we will test conditioned media from rat epicardial cells using our neonatal and adult proliferation assays. Finally, we will examine extracts and conditioned media isolated from fetal and neonatal cardiac myocytes. These Specific Aims will form the basis of a subsequent Phase II application focused on purification and cloning of a specific cardiac mitogen, which will then be used for pre-clinical/clinical studies. Because a molecular approach to cardiac regeneration in industry is unique and addresses an enormous unmet medical need, Hydra Biosciences successfully completed a series A financing of $10.6 million